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Targeting Bacterial Gyrase with Cystobactamid, Fluoroquinolone, and Aminocoumarin Antibiotics Induces Distinct Molecular Signatures in Pseudomonas aeruginosa.

ABSTRACT: The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins, and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at subinhibitory concentrations could be distinguished clearly by RNA sequencing as well as metabolomics. We observed a strong (2.8- to 212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with extracellular DNA (eDNA) release. Gyrase B-binding aminocoumarins induced the most pronounced changes, including a strong downregulation of phenazine and rhamnolipid virulence factors. Cystobactamids led to a downregulation of a glucose catabolism pathway. The study implies that clustering cellular mechanisms of action according to the primary target needs to take class-dependent variances into account. IMPORTANCE Novel antibiotics are urgently needed to tackle the growing worldwide problem of antimicrobial resistance. Bacterial pathogens possess few privileged targets for a successful therapy: the majority of existing antibiotics as well as current candidates in development target the complex bacterial machinery for cell wall synthesis, protein synthesis, or DNA replication. An important mechanistic question addressed by this study is whether inhibiting such a complex target at different sites with different compounds has similar or differentiated cellular consequences. Using transcriptomics and metabolomics, we demonstrate that three different classes of gyrase inhibitors can be distinguished by their molecular signatures in P. aeruginosa. We describe the cellular effects of a promising, recently identified gyrase inhibitor class, the cystobactamids, in comparison to those of the established gyrase A-binding fluoroquinolones and the gyrase B-binding aminocoumarins. The study results have implications for mode-of-action discovery approaches based on target-specific reference compounds, as they highlight the intraclass variability of cellular compound effects.

SUBMITTER: Franke R

PROVIDER: S-EPMC8407119 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bacteria frequently encounter selection by both phages and antibiotics. However, our knowledge on the evolutionary interactions between phages and antibiotics are still limited. Here, we characterized a phage-resistant Pseudomonas aeruginosa variant PAO1-R1 that shows increased sensitivity to gentamicin and polymyxin B. Using whole genome sequencing, significant genome differences were observed between the reference P. aeruginosa PAO1 and PAO1-R1. Compared to PAO1, 64 gene-encoding proteins with nonsynonymous single nucleotide polymorphisms (SNPs) and 31 genes with insertion/deletion (indel) mutations were found in PAO1-R1. We observed a significant reduction in phage adsorption rate for both phage vB_Pae_QDWS and vB_Pae_W3 against PAO1-R1 and proposed that disruption of phage adsorption is likely the main cause for evolving resistance. Because the majority of spontaneous mutations are closely related to membrane components, alterations in the cell envelope may explain the antibiotic-sensitive phenotype of PAO1-R1. Collectively, we demonstrate that the evolution of phage resistance comes with fitness defects resulting in antibiotic sensitization. Our finding provides new insights into the evolutionary interactions between resistance to the phage and sensitivity to antibiotics, which may have implications for the future clinical use of steering in phage therapies. IMPORTANCE Bacteria frequently encounter the selection pressure from both antibiotics and lytic phages. Little is known about the evolutionary interactions between antibiotics and phages. Our study provides new insights into the trade-off mechanism between resistance to the phage and sensitivity to antibiotics. This evolutionary trade-off is not dependent on the outer membrane proteins (OMPs) of the multidrug efflux pumps. The disruption of phage adsorption that induced phage resistance and the changes in structure or composition of membranes are presumably one of the major causes for antibiotic sensitivity. Our finding may fill some gaps in the field of phage-host interplay and have implications for the future clinical use of steering in phage therapies.

Dataset Information

Targeting Bacterial Gyrase with Cystobactamid, Fluoroquinolone, and Aminocoumarin Antibiotics Induces Distinct Molecular Signatures in Pseudomonas aeruginosa.

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